P53 Mutation in Inflammatory Breast Cancer Cell Lines.

Abstract

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary breast cancer and represents 1% to 2% of primary breast cancer in the United States. IBC is characterized by an acute inflammation of the skin of the affected breast generally believed to be caused by blockage of the dermal lymphatics by tumor emboli. Wild type (WT) p53 is a tumor suppressor gene, which induces apoptosis and p53 mutations are associated with poor prognosis in breast cancer. Compared with locally advanced breast cancers, IBC patients have higher levels of mutated p53 protein that has been associated with more aggressive tumors, anthracycline resistance, shorter progression free survival, shorter overall survival, and less favorable long-term outcome. The aim of this study was to determine if there are unique genetic variations in IBC cell lines that would provide specific genetic p53 mutations that could be exploited for targeted therapy with the intent of improving response to treatment and overall survival in IBC.Materials and Methods: Genomic DNA was extracted from six breast cancer cell lines (MDA-453, SUM149, MCF-7, KPL4, MDA321, and SUM190) and the immortalized human mammary epithelial cells (HMLE) using the Qiagen DNA Blood Mini Kit (Valencia, CA). Among the 6 breast cancer cell lines, KPL-4, SUM149 and SUM190 are IBC cell lines; MDA231 and SUM149 have basal-like phenotype; MCF-7 has wild-type p53; SUM190 and KPL-4 are Her2 amplified. The DNA purity and concentration were determined by spectrophotometric measurements of absorbance at 260nm and 280 nm. Polymerase chainreaction (PCR) was performed to amplify the fragments of exons 2-11 of the p53gene using consensus primers. The PCR products were scanned and identified using the Agilent Bioanalyzer 2100. DNA sequencing was performed on PCR products in the ABI PRISM 310 Genetic Analyzer. The BLAST search was used to identify p53 mutations compared with the reference sequence, X54156, from Genbank.Results: We screened 2-11 exon sequences of the p53 gene in the 7 human breast cell lines. We identified two IBC cell lines (SUM149, SUM-190) with a p53 gene alteration that predicted a change in the encoded protein, SUM149 at exon 7 (ATG to ATA, Met-237-Ile) and SUM190 at exon 9 (CAG to TAG, Gln -317-stop). Both mutations have been previously reported. Five nonsense mutations were identified in two other cell lines, MDA-453 and MDA321. No mutations were identified in KPL4 and HMLE cells.Conclusions: The p53 mutation profile in breast cancer cell lines suggests an additional biological feature for the characterization of IBC. Furthermore, these data support the previously reported association between p53 status and chemo- and radioresistance in this disease responsible for poor prognosis. Therapies directed to restore p53 function should be explored in IBC models and in clinical trials. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3161.